Recent advances in genomic DNA sequencing of microbial species from single cells

Abstract

The vast majority of microbial species remain uncultivated and, until recently, about half of all known bacterial phyla were identified only from their 16S ribosomal RNA gene sequence. With the advent of single-cell sequencing, genomes of uncultivated species are rapidly filling in unsequenced branches of the microbial phylogenetic tree. The wealth of new insights gained from these previously inaccessible groups is providing a deeper understanding of their basic biology, taxonomy and evolution, as well as their diverse roles in environmental ecosystems and human health.

Figure 1. Complementary methods used to investigate the genomics of uncultivated bacteria

a | PCR amplification of the 16S ribosomal RNA gene can be carried out from most novel bacteria using primers that anneal to sequences which are highly conserved across bacterial species. Variable regions of the 16S rRNA gene can then be used to derive a phylogenetic tree. b | Metagenomics is based on sequencing of total DNA extracted from the environment. Gene content and frequencies are obtained for the entire ecosystem; however, assembly of the sequencing reads from the sequencing library inserts (which are indicated by coloured sequences) into the genomes of individual species is complicated by the large number of organisms that contribute to the DNA in the sample. c | Single-cell sequencing typically does not recover the entire genome; however, the reads obtained in the sequencing library are all genetically linked, which facilitates genome assembly. These genomes represent the integrated genetics and biochemistry of individual microbial species. Single-cell genomes can serve as a reference genome to aid the assembly of sequencing data for uncultivated species that are closely related. DNA or cDNA sequencing reads obtained through shotgun metagenomic or metatranscriptomic approaches can be mapped to the reference genomes (dashed arrow). Single-cell reference genomes can also provide phylogenetic placement for a substantial portion of the metagenomic reads that have not been previously assigned a taxonomy. 16S rRNA gene PCR is a rapid and inexpensive method to assign phylogeny and community composition. Metagenomics also provides community composition and gene content. Single-cell sequencing provides assembled contigs to determine genes and pathways within an individual cell and is best suited for the evaluation of strain variations. MDA, multiple displacement amplification.

Figure 2. Filling in the bacterial tree of life

The 16S ribosomal RNA gene maximum likelihood phylogenetic inference for the domain Bacteria highlights the uncultivated phyla and the impact of single-cell genomics. Of 61 known phyla in the bacterial domain, 32 still have no cultivated representatives (red branches). The majority of genomes from the uncultivated candidate phyla were captured using multiple displacement amplification (MDA)-based single-cell approaches (green dot). Sequences are grouped on the phylum level, except for the phylum Proteobacteria, which is polyphyletic in this analysis. Groups containing 16S rRNA genes from cultured isolates are in blue. The scale bar represents 10% estimated sequence divergence.

Figure 3. Comparative genomics using single-cell DNA amplification

Amplification of DNA by multiple displacement amplification (MDA) can achieve single-nucleotide resolution in resequencing studies (that is, studies in which a known reference genome is available) for use in analyses of single-nucleotide polymorphisms (SNPs), insertions and deletions (indels), and structural variations, as well as in whole-genome comparisons from de novo assembled sequences. A | SNP and indel analyses have been carried out on a virulence-related gene (the reference sequence is shown at the top of each panel) of Porphyromonas gingivalis for three independent amplified single cells (MDA1, MDA2 and MDA3) that were captured from a complex biofilm in a hospital, which revealed shared variants (that is, SNPs) within this gene. B | With advances in MDA-optimized assembly tools such as SPAdes, the technique is approaching the level expected for sequencing cultured strains, which is improving whole-genome comparisons of synteny (that is, the order of genes in a genome) (part Ba; syntenic blocks that are shared between genomic regions are connected with coloured ribbons). Similarly, this technique can improve gene discovery (part Bb). In this example, an assembled draft genome (JCVI SC001), which consists of the SPAdes assembly of MDA3, is compared against reference genomes from the P. gingivalis strains TDC60, W83 and ATCC 33277 (REF. 25). De novo assembly of regions that contain multiple repeats and that are difficult to assemble, such as the clustered regularly interspaced short palindromic repeat (CRISPR) region, can be resolved (part Bc). For example, de novo assembly of the repeats in CRISPR region 36–30 showed that the repeat regions in MDA3 were identical to sequenced genomes from cultured pathogen isolates (W83 and TDC60) but contained variable spacer sequences, which is indicative of phage predation in a different environment. megL, methionine gamma-lyase. Parts A and Bc are adapted with permission from REF. , Cold Spring Harbor Laboratory Press.